The major objective of this project is to elucidate the mechanism by which transcription is controlled in eukaryotic cells. Emphasis is placed on the direct role played by multiple DNA-dependent RNA polymerases and ancillary chromosomal and nonchromosomal proteins which may affect the polymerases. The approach used is to reconstruct an in vitro transcription system for isolated genes (ribosomal RNA genes) and to compare factors affecting that system with changes in in vivo transcription of the same genes during differentiation of a simple protozoan, Acanthamoeba castellanii. Polymerases purified by bulk protein methods or by immunoprecipitation from several stages of synchronous encystment of acanthamoeba will be examined for 1. addition, deletion or 2. covalent modification of subunits and for 3. changes in specificity in vitro. Acanthamoeba extracts will be screened for proteins stimulating transcription in vitro in a transcription system utilizing isolated rRNA genes for template. Recombinant DNA techniques will be utilized to produce the DNA template. Specificity will be determined by hybridization competition, symmetry tests and initiation sequence determination. The mechanism of any isolated stimulatory factor or core modification will be determined by DNA-RNA polymerase binding studies. Transcription of isolated chromatin and chromatin reconstituted from purified chromosomal proteins and recombinant DNA will be examined to determine the role of chromatin structure in transcriptional specificity.